The resistance to IPC in mutant N. sylvestris lines were confirmed by us by series of tests and morphocytochemical analyses. For instance, the morphocytochemical analysis of control and mutant lines revealed 6–8 times more resistance to IPC of mutant line as compared with control one. It was found also that the increasing doses of IPC lead to dramatic changes in nucleus and microtubule network in sensitive plants. In control plants tripolar anaphases and telophases were found after treatment with 30 MkM IPC. IPC-treatment resulted also in high number of metaphase+anaphase (from 32.48% in control cells without treatment to 80.07% after treatment) and accumulation of multinuclear cells and lobbed nuclei in treated control cells (see Figure 1d–f).
Threshold level of IPC induced an apoptotic process, characterised by cell cycle arrest, hypercondensation of chromatin, an appearance of lobbed nuclei; abnormal, multipolar mitotic spindles, post-mitotic micronuclei formation due to nuclei fragmentation in the root cells of sensitive lines. However, root meristematic cells from resistant plants revealed no disturbances in cell cycle progression after treatment with 30 MkM IPC (see Figure 1a–c).
Further investigations shown that control and mutant N. sylvestris plants possessed some morphological differences (see Figure 2). Mutant plants characterised in certain depression resulted in reduction of sizes of vegetative and generative organs, and of plant bodies on the whole. There were slight differences in the appearance of flowers (see Figure 3a) and more visible differences in the length of stamens and pistils between control and mutant lines (see Figures 3b and 3c).
More detailed analysis of reproductive organs development of IPC-resistant mutants revealed that microspores in anthers are subjected to degenerative process, leading to pollen sterility. It was found also the chromatin agglutination in nuclei of most part of microsporocytes.
During study the microsporogenesis in IPC-mutant plants it was revealed the formation of microspores of different sizes (gigantic, medium and dwarfic: 15.05 ± 0.06 mkm, 7.50 ± 0.23 mkm and 5.15 ± 0.13 mkm in size, respectively) and in different numbers (dyads, triads and polyads). In the same time normal tetrads produced in control N. sylvestris plants (data not shown). Variation in sizes and numbers indicates on disturbances in chromosome separation in I and II meiotic divisions, loss the second division in one (in the case of triads) and in both dyads, the formation of multipolar spindles (in the case of polyads) in mutants.
It was established that pollen grains of control plants possessed the high level of fertility (97.12 ± 0.33%); they were homogenous and synchronous in development. Whereas, genetically non-balanced microscopes of mutant lines after tetrad dissipation were unable to further development. They did not synthesise the fats and new cytoplasm and underwent typical cell death. Only 14.73 ± 1.09% formed microspores got down later to division with vegetative and generative cells formation.
As a result, the common number of pollen was reduced in anthers of mutant plants. About 2.88 ± 0.33% of bicellular pollen grains were hydrated and stopped in development. Consequently, the mature pollen of mutant plants was morphologically and functionally non-homogenous. Its fertility in different flowers was from 7.63 to 27.93%. Sterile pollen grains were small, weakly stained or non-stained after incubation with dye (see Figure 5b). They also had reduced contents of cytoplasm as compared with control plants.
The results of female gametophyte study in mutant and control N. sylvestris plants demonstrated that ovary development after pollination was lower in 1.5–2 times in mutant plants as compare with control ones (see Figure 6). The delay in growth and development was connected with the high number of non-fertilized bud seeds (see Figure 7) in mutant plants. We found strong correlation between fertility level of pollen and a number of fertilized bud seeds in these plants.
Analysis of fertilized bud seeds of N. sylvestris mutants shown that bud seeds were different in the sizes that indicates on their non-homogeneous and asynchronous development as compare with control plants. It was found that a speed of embryos development in mutant plants was lower than that in control lines, and sometimes an embryo development stopped on early globular stage. Therefore, the seed production in mutant plants was very low due to both high pollen sterility and decreased viability of bud seeds development.